pPZP211

Producing vaccines in plant cells,known as phytopharmaceuticals or molecular agriculture,is a promising large-scale production technology.Here,researchers report the stable expression of the amino acid sequence corresponding to the Mycoplasma gallisepticum TM-1 gene as a candidate vaccine antigen against Chronic Respiratory Disease(CRD)in chickens using wheat seed′s tissues as a production host.Molecular and immunoblot analyzes confirmed that the recombinant 41.8-kDa protein was ubiquitously expressed in endosperm tissue at an expression level of 1.03 mg/g dry weight.When administered orally,the plant-made vaccine was effective in generating an antibody response in an animal model(i.e.,chickens)without any detectable weight loss.The research shows that plant-based vaccines are not only safe,but also scalable and cost-effective,with long-term stability at room temperature.

In this study,the binary vector pPZP211 vector was purchased commercially(Creative Biogene).The rice endosperm-specific promoter GluB-4 and Nos terminator were inserted upstream and downstream of the EcoRI/KpnI and XbaI/HindIII sites of the multiple cloning site(MCS),respectively.The codon-optimized complete CDS of the Mycoplasma gallisepticum TM-1 protein is followed by the endoplasmic reticulum retention signal KDEL(Lys-Asp-Glu-Leu)attached to the TM-1 C terminus.The CTB(cholera toxin B subunit)coding sequence followed by a furin cleavage site(RRKRSV)was inserted immediately upstream of TM-1.The entire PGluB-4-CTB-furin-TM1-KDEL-Tnos cassette was synthesized and subcloned into the KpnI/XbaI sites in pPZP211.

Figure 1.A.pPZP211:Schematic diagram of CTB-TM-1 expression cassette.(The pPZP211 vector was purchased from Creative Biogene.)B.Characterization of the genomic integration of the pPZP211:CTB-TM-1 cassette in different transgenic lines by PCR using primers(CTB-F and TM-1-R).C.Verification of expression of recombinant CTB-TM-1 in three lines by Western blotting using anti-CTB antibody.D.Quantification of recombinant CTB-TM-1 expression in C.E.GM1 ELISA detects the folding of CTB-TM-1 and its binding to the GM1 receptor.